The introduction presented in this chapter is in part published in: Rast, M.I., and Simon, R. (2008). The meristem-to-organ boundary: more than an extremity of anything. Curr Opin Genet Dev 18, 287-294. 1 CHAPTER I INTRODUCTION

1. INTRODUCTION

1.1. Embryonic pattern formation Plant development proceeds in a different manner to that of animals, as plant organogenesis occurs postembryonically through the activity of the shoot apical meristem (SAM) and the root meristem (RM). All above ground tissues such as leaves, flowers and shoot branches ultimately derive from the SAM. The root system, consisting of primary and secondary roots, derives from the RM. Nevertheless, establishment of the two apical meristems, formation of the apical-basal and radial axis, as well as determination of the basic plant body requires a precise order of cell divisions during plant embryogenesis. This process, termed embryonic pattern formation, is therefore fundamental for further postembryonic growth and develop-ment. Embryonic pattern formation starts with an asymmetric division of the zygote that produces a smaller apical (ac) and a larger basal cell (bc) (Fig. 1.1). The apical daughter cell under-goes several stereotypical cell divisions to give rise to the proembryo. The basal daughter cell divides to generate the suspensor which serves as a connection between the developing embryo and maternal tissue. Only the uppermost suspensor cell, the hypophysis (hy), adopts an embryonic fate. At globular stage this cell undergoes a sequence of reproducible divisions to give rise to the quiescent centre (QC), the future organizer of the RM (Scheres et al., 1994). Further refinement of the embryonic pattern occurs during succeeding developmental stages. Finally, the mature embryo consists of four distinct structures: cotyledons, SAM, hy-pocotyl and root harboring the RM (Fig. 1.1; reviewed in Moller et al., 2009)

Fig. 1.1: Stages of embryo development. The zygote divides asymmetrically to produce a smaller apical (ac) and a larger basal cell (bc). Descendants of the apical daughter cell undergo a sequence of reproducible cell divisions to give rise to the proembryo. The basal daughter cell divides transversally to produce the extra-embryonic suspensor. At globular stage the uppermost suspensor cell becomes specified as hypophysis (hy) and contributes to the embryonic RM. Colors identify origins of the five structures of mature embryos. 2 CHAPTER I INTRODUCTION

1.2. Organization of the Shoot Apical Meristem (SAM) The dome shaped SAM can be subdivided into different zones and layers on the basis of cell division rate and orientation, cell origin and morphology (Fig 1.2). The central zone (CZ) con-tains slowly dividing pluripotent stem cells. Their daughter cells are displaced to the sur-rounding peripheral zone (PZ). Cells in the PZ divide more rapidly and can join each other to found new organs and enter the pathway towards differentiation (Steeves et al., 1989). To separate organ founder cells and stem cell descendants in the PZ, morphological bounda-ries, consisting of distinct, mitotically nearly inactive cells, are formed (Kwiatkowska, 2006; Breuil-Broyer et al., 2004; Aida et al., 2006). The rib meristem beneath the CZ and PZ gives rise to the plants corpus and vasculature. In a longitudinal section, the SAM is composed of three clonally distinct cell layers (L1-L3; Fig. 1.2B). Cells in the L1 and L2 preferentially divide anticlinal; thus, their daughter cells remain in their layer of origin. The L1 layer consists of epidermal progenitors, while cells in the L2 will give rise to sub-epidermal tissues and the gametes. The multilayered L3 shows anti- and periclinal cell divisions and produces the majority of the plants ground tissue and vasculature (Vaughn, 1952; Steeves, 1989). As stem cells are located in the upper 4-5 cell layers of the CZ, they contain cells of all three clonal layers. The organizing centre (OC), a group of cells with a low division rate beneath the CZ, is required for the initiation of stem cells during embryogenesis and later for their maintenance (reviewed in Bleckmann et al., 2009).

Fig. 1.2: SAM organization. (A) Scanning Electron Micrograph (SEM) of an Arabidopsis SAM. The central zone (CZ; yellow) at the summit of the meristem contains slowly dividing stem cells; stem cell descendants are shifted (arrows) to the peripheral zone (PZ) where they form new organ primordia (P1; P2) or contribute to the boundary formation (dark blue). After floral transition, determinate floral meristems (FM) are initiated at the SAM flanks. (B) The SAM consists of three clonally distinct cell layers (L1, L2 and L3). In the L1 and L2 layer cell divisions are preferentiallly anticlinal, cell divisions in the L3 occur in all planes. The stem cell population in the CZ (yellow) contains cells of all three layers. The organizing centre (OZ, red) is required for stem cell maintenance. Modified from Bleckmann et al., 2009.

3 CHAPTER I INTRODUCTION

During the vegetative stage, the SAM produces only rosette leaves. After floral transition, new, specialized meristems, that will produce shoots (axillary meristem (AXM)) or flowers (floral meristem (FM)), are initiated in the PZ. Each FM establishes floral organs in four con-centric whorls: 4 sepals, 4 petals, 6 stamen and 2 carpels. The FM, in contrary to the SAM, is determinate: it arrests after it produced the full range of floral organs.

1.2.1. SAM homeostasis and lateral organ formation Genetic mosaics and laser ablation experiments showed that a cells position within the SAM and not its clonal origin determines its fate (Poethig, 1989, Irish, 1991, Reinhardt et al., 2003). Indeterminate shoot growth therefore requires a balance between stem cell division and daughter cell differentiation to maintain the domain specific SAM organization (Fig. 1.2). Within the past years, genetic analyses have identified a number of transcriptional regula-tors required to control meristem homeostasis and organ development. The analyses of mu-tant phenotypes and expression studies of the corresponding genes have shown that a mu-tual downregulation between meristem specific and organ specific genes is essential for normal development. Moreover, several studies highlighted the role of boundary establish-ment between the meristem and organ primordia. It was shown that cells within these boundaries play dual roles. A number of transcriptional regulators encoded by boundary specific genes act to repress cell division and growth, resulting in the separation of organs from the meristem (M-O boundaries) or in a separation of adjacent organs (O-O boundaries) (Breuil-Broyer et al., 2004; Aida et al., 2006; Kwiatkowska, 2006). Beside this function, boundary specific genes participate in various regulatory networks to define and maintain indeterminate and determi-nate cell fates in the SAM (reviewed in Aida et al., 2006). More detailed information about the regulatory networks, involving meristem, organ and boundary specific genes, are provided in the enclosed review (Rast et al., 2008).

1.2.2. The meristem-to-organ boundary: more than an extremity of anything The review: “The meristem-to-organ boundary: more than an extremity of anything” (Rast et al., 2008) was published in Current Opinon in Genetics and Development (impact factor: 9.3). The manuscript was written by me and overworked by Prof. Dr. R. Simon.

4 Available online at www.sciencedirect.comThe meristem-to-organ boundary: more than an extremity ofanythingMadlen I Rast and Ru¨diger SimonIn plant shoot meristems, cells with indeterminate fate are with distinct gene expression programs and behavior. Inseparated from determinate organ founder cells by this review, we will ﬁrst discuss how sites of organmorphologicalboundaries.Organcellsareselectedat formation are selected, which gene expression programssites of auxin accumulation. Auxin is channeled between cells are involved, and then concentrate on the functions andvia efﬂux carrier proteins, but inﬂux carriers are needed to interactionsofgenesthatareexpressedspeciﬁcallyintheconcentrate auxin in the outer meristem layer. The genetic meristem-to-organ boundary.programmes executed by organs and meristems areestablished by mutual repression of transcription factors, Where organs are made: a primer oninvolving the sequestration of enhancer elements into DNA phyllotaxisloops. Boundary cells play a dual role in separating and In most plants, organs are initiated at regular angles tomaintainingmeristemandorgandomains,andexpressunique each other. The groundlaying mechanism for generatinggenes that reduce cell division and auxin efﬂux carrier activity, suchphyllotacticpatternsinvolvesthetransportandlocalbutactivatemeristematicgeneexpression.Boundarypositions accumulation of the phytohormone auxin. Owing to adepend on signals emitted from indeterminate cells at the lowerextracellularpH,auxinisunchargedwhenenteringmeristem center. the cell, but becomes deprotonated inside and requiresthe help of membrane resident auxin export carriers toAddress leavethecellagain.AkeymoleculeisPIN1[1],anauxinInstitute of Genetics, Heinrich Heine University, D-40225 Du¨sseldorf, efﬂux carrier that is predicted to be oriented in cell wallsGermany towardsthehigherauxinconcentration,therebypumpingauxin against aconcentration gradient[2 ].Using repor-Correspondingauthor:Rast,MadlenI.(madlen.rast@uni-duesseldorf.de)tergenesthataresensitivetoauxinsignaling,ithasbeenand Simon, Ru¨diger (ruediger.simon@uni-duesseldorf.de)shown that auxin accumulates at sites of future organinitiation,andissimultaneouslydepletedfromcellsintheCurrent Opinion in Genetics & Development 2008, 18:287–294 vicinity [3,4]( Figure 1). Within the developing leafprimordium, auxin is then channeled towards the stemThis review comes from a themed issue onPattern formation and developmental mechanisms tissue below. Where auxin levels are artiﬁcially raised atEdited by Ottoline Leyser and Olivier Pourquie´ theﬂankofameristem,aneworganwillbegenerated[5].By following a set of simple rules governing auxin diffu-Available online 14th July 2008sion and the activity and orientation of auxin efﬂux0959-437X/$ – see front matter carriers, virtual meristem models can be generated that# 2008 Elsevier Ltd. All rights reserved. allow in silico reproduction of the phyllotactic patternsobservedinnature[4,6].Inthesesimulations, onlyauxinDOI 10.1016/j.gde.2008.05.005distribution in the outer meristem layers is considered tocontrol patterning.Introduction However, extracellular (apoplastic) auxin could get lostMosthigherplantsmaintaintheuniqueabilitytoproduce from this patterning engine by diffusion, and it has beennew organs throughout their entire lifetime. This is proposed that also auxin inﬂux carriers of the AUX1/possible owing to collections of pluripotent cells called LAX1 family are redundantly required to maintain highthe shoot apical meristem (SAM) that reside at the shoot auxin concentrations in the outermost layer of the mer-

tip. The dome-shaped SAM carries non-differentiating istem[7 ].Eliminatingall auxin inﬂux carrier activityinstem cells at its top region, which divide slowly to gen- quadruple mutant combinations severely disturbs phyl-eratemorecellsasthebuildingmaterialforlateralorgans. lotaxis, causing the formation of primordia at irregularWhen stem cells divide, daughter cells are shifted out- angles, or even primordia cluster, because sharp auxinwards to the periphery, where they can join others to peaks cannot be maintained. But in contrast to pin1found a new organ, or differentiate after further division mutants that also show organ fusions and alterations inrounds.Cellfateisthereforeconnectedtoacell’slocation organsize,theinter-organboundariesarestillestablished,within the meristematic dome. Between meristematic which becomes evident from the formation of separatecellsandtheyoungorgan,aboundaryisgeneratedwhich organs. Once initiated, a primordium could generate anisnotonly‘...thatwhichisanextremityofanything...’, inhibitory ﬁeld that prevents the formation of furtherasEucliddeﬁnedit;instead,itconsistsofspecializedcells primordia in the immediate vicinity. Because pin1www.sciencedirect.com Current Opinion in Genetics & Development 2008, 18:287–294